Cancer metastasis, not the primary tumor, is the main cause of death from breast cancer. To metastasize, a cancer cell must leave the primary tumor, survive in blood or lymphatic circulation, invade and colonize distant tissues and establish metastatic foci. Most carcinomas are thought to disseminate via the blood stream and the lymphatic system in parallel. Lymph node metastases are thought to arise from tumor cells that have entered the natural lymphatic drainage of the breast tissue to colonize the downstream sentinel nodes. Beyond this general level of understanding about lymphatic metastasis, there are fundamental biologic questions about lymphatic dissemination that remain unanswered such as: How do tumor cells enter the lymphatic drainage? What is the efficacy of lymphatic metastasis in vivo? Do metastatic and non-metastatic tumors both shed cells into the lymphatic drainage? Are the tumor cells that enter the lymphatic drainage a specialized subset of the parental tumor population? What specialized molecular mechanisms do these cells use to enter the lymph, thrive in the lymph fluid, and colonize the lymph node? To answer these questions, first, our laboratory has developed an innovative microsurgical technology to collect lymph, and the lymph circulating tumor cells (LCTC) contained therein, in an immunocompetent breast cancer animal model. Subsequently, in a collaborative effort with medical surgeons in our community, and those in Indiana University School of Medicine, we successfully applied this same technology to collect, for the first time, LCTC entering the lymph drainage in women with metastatic breast cancer. Second, we propose that the Toll-Like Receptor (TLR) signaling pathways is a major molecular determinant of tumor cell entry into the lymphatic drainage. TLR signaling is known to regulate pathogen sensing and homing of immune cells. This pathway also regulates cytokine and lymphokine release that are part of lymph node activation during the early stages of infection. In the present proposal we will evaluate expression levels of TLRs and key proteins associated with TLR cell signaling pathways for the first time in breast cancer cells (LCTC) entering the lymphatic drainage in the rat model of breast cancer as well as in women with breast cancer. We will use these unique cells (LCTC) to evaluate the functional role of activating or suppressing the TLR pathway on the extent of lymph node metastasis in the rodent model. In women with breast cancer we will correlate expression levels of TLRs and proteins associated with TLR signaling pathways in LCTC with the extent of metastasis in the sentinel node. Understanding the factors that contribute to the first step of lymph node metastasis can lead to a new class of therapies that suppress dissemination, essentially turning a malignant tumor into a benign tumor. Direct access to the tumor cells entering the lymph circulation provides a completely novel paradigm for prognosis and individualized therapy.